Tumor necrosis factor (TNF) was first discovered as a tumor cytotoxin that preferentially killed growing tumor cells. After cloning of the gene and purification of recombinant protein, it soon became clear that TNF also exerts biological effects on different normal cell types. The therapeutic value of TNF in treatment of cancer is limited by toxic side effects occurring at high TNF dose, and by a wide variation in TNF sensitivity of tumor cells. Understanding of the molecular signaling pathways leading to TNF cytotoxcity can greatly contribute to our search for strategies to improve the therapeutic value of TNF. Although considerable effort has been devoted to clarify the post-receptor mechanisms of TNF, the available data are difficult to interpret the TNF-mediated cytotoxcity to tumor cells. The primary long-term objective of this project is to elucidate the intracellular signaling and molecular mechanisms of TNF-mediated cytotoxcity. Our specific aims will be: (1). To search for the downstream targets of FADD and new proteins involved in dimerization of death domain by Yeast two hybrid assay. (2). To identify the mediators which render tumor cells sensitive or resistant to the cytotoxic action of TNF. This will be accomplished by using random inactivation of genes via the introduction of cDNA expression libraries to identify the relevant genes.